Jones, L.T.

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Investigation of cholecalciferol-dependent protein biosynthesis in the cytosol of the small intestine of the rachitic chick 

Volume 108, number 1 FEBS LETTERS December 1979 
INVESTIGATION OF CHOLECALCIFEROL-DEPENDENT PROTEIN BIOSYNTHESIS IN 
THE CYTOSOL OF THE SMALL INTESTINE OF THE RACHITIC CHICK 
L. T. JONES* 
Dunn Nutritional Laboratory, University of Cambridge and Medical Research Council, Milton Road, Cambridge CB4 IXJ, England 
Received 8 October 1979 
1. Introduction 
Definite progress with regard to the discovery of 
the exact nature of the biosynthesis of the chole- 
calciferoldependent calcium-binding protein (CaBP) 
has been made [ 1,2 1. It has been demonstrated that 
the active metabolite of cholecalciferql, 1,25dihydroxy- 
cholecalciferol gives rise to a specific mRNA which 
induces synthesis of one or more cellular proteins 
concerned with the movement of calcium across the 
cell. This investigation has demonstrated that 3 chole- 
calciferol-dependent protein components are detect- 
able, one of which is CaBP. 
2. Materials and methods 
2 .I . Animals 
One day old Rhode Island X Light Sussex chicks 
were obtained from the National Institute for Research 
in Dairying, Shinfield, Reading. The chicks were used 
when they were 4 weeks old having been fed upon 
the rachitogenic diet described [3]. 
2.2. Chemicals. 
Crystalline cholecalciferol (activated 7dehydrocho- 
lesterol) was obtained from Peboc Limited, Middlesex. 
Prior to intracardial injection propylene glycol was 
added to an ethanolic solution of cholecalciferol such 
that the ratio of propylene glycol to ethanol was 9: 1 
* Present address: Division of Nutrition and Food Science, 
Department of Biochemistry, University of Surrey, 
Guildford, England 
ElsevierlNorth-Holkmd Biomedical Press 
(v/v), and 0.1 ml was used. The radioactive amino 
acid leucine was obtained from the Radiochemical 
Centre,Amersham as L-[ 1 -14C]leucine and L-[4,5-3H]- 
leucine and was made up in 0.9% NaCl before intra- 
cardial injection of 0.1 ml. 
2.3. Tissue preparation 
These double-isotope-labeling experiments involved 
the use of [14C]leucine and [3H]leucine in groups of 
3 chicks, dosed and undosed, with cholecalciferol 
consecutively at 4 separate time intervals. Thus 12 
chicks fed upon the diet mentioned earlier from 1 day 
old were injected intracardially at 4 weeks of age 
with 5 pg cholecalciferol. Three chicks were killed 
4,8, 12 and 24 h after cholecalciferol administration, 
these chicks having also received 40 &i [14C]leucine 
injected intracardially 1.5 h before slaughter. A fur- 
ther 12 rachitic chicks undosed with cholecalciferol 
were killed simultaneously, 3 at each time-interval 
having been injected with 60 PCi [3H]leucine 1.5 h 
before. The lirst 15 cm of small intestine following 
the pylorus wasexcised,washed through with ice-cold 
0.9% NaCl, slit longitudinally and blotted. The 
mucosal tissue was scraped off using a spatula and 
homogenised in buffer (pH 7.4) (20%, w/v) with a 
Potter-Elvejhem homogeniser and Teflon pestle. The 
composition of the buffer used was 0.0127 M Tris, 
0.12 M NaCl, 4.74 mM KC1 and 1 mM mercapto- 
ethanol adjusted to pH 7.4 with N HCl [3]. The 
homogenate was spun at 105 000 X g for 1 h in an 
MSE Superspeed centrifuge to provide the soluble 
fraction of the cell. These fractions from each group 
here mixed at the same time interval so that the 
number of 14C and ‘H dpm values were the same. 
17 
Volume 108, number 1 FEBS LETTERS December 1979 
2.4. Polyacrylamide disc gel electrophoresis, gel 
slicing and radioactive counting 
After mixing the cytosol samples from both 
cholecalciferol-dosed and undosed groups in the ratio 
1: 1 (v/v) 500 ~1 were applied to 15% polyacrylamide 
gels. Composition of the solutions and the methodology 
used during electrophoresis are given in [4]. Duplicate 
gels were run for each time interval, one gel was 
stained and the other was frozen in dry-ice before 
being sliced into 1 mm slices using a macrotome 
GTS-1 (Yeda Ltd., Rehovot). These slices were placed 
30 
0 
$2.0 
oz 
K 
2 
1.0 
2.0 
0 1.0 
t 
a 
I 0 
3 
: 
4 HOUR TIME INTERVAL 
in scintillation vials with 1 ml 90% NCS in water and 
incubated in an oven at 60°C for 3 h. The vials were 
cooled and 10 ml scintillation fluid [4] was added for 
radioactive counting in a Packard Tri-Carb Automatic 
Liquid Scintillation Counter. 
3. Results 
Figure 1 indicates the distribution of radioactive 
amino acid incorporation into chick cytosol protein 
3.07 
I 8 HOUR TIME INTERVAL 
a 
I 
i 2 i i 5 
cm. along gel + 
12 HOUR TIME INTERVAL 
cm. along gel + 
2.0 
0 i i i i ’ 
cm. along gel : 
24 HOUR TIME INTERVAL 
0 1 2 3 4 
cm. along gel + 
Fig.1. Effect of cholecalciferol on the distribution of labelled leucine incorporation (14C/“H) into chick cytosol protein at various 
intervals using polyacrylamide disc gel electrophoresis. 
18 
Volume 108, number 1 FEBS LETTERS December 1979 
at the 4 time intervals studied. At the first time inter- 
val of 4 h, only 2 radioactive peaks are observed 
indicating the presence of 2 cholecalciferol-dependent 
protein components, none of which migrates at the 
same rate as the prepared sample of chick intestinal 
CaBP [3] run in parallel. At the remaining 8,12 and 
24 h time intervals 3 cholecalciferoldependent pro- 
tein components are detected one of which migrates 
at the same rate as the cholecalciferoldependent 
CaBP. 
4. Discussion 
The presence of 2 cholecalciferol-dependent pro- 
tein components 4 h after injection of cholecalciferol 
could be interpreted as meaning either that they are 
both association and dissociation products of CaBP or 
that protein biosynthesis other than CaBP has been 
detected. The other 3 time intervals following injec- 
tion with cholecalciferol indicate the presence of 
2 components as well as a third component which 
migrates at the same mobility as CaBP. It is interest- 
ing to note that it was observed [.5] that following 
prolonged storage, purified bovine intestinal CaBP 
gives rise to 2 further protein components, i.e ., a 
total of 3 components. This study has shown the 
detection of 3 cholecalciferol-dependent protein 
components present shortly after dosage of the 
rachitic chick with cholecalciferol. It is a possibility 
therefore that the 3 components found in this work 
using polyacrylamide gel electrophoresis are all sub- 
units of CaBP. 
Acknowledgement 
I acknowledge the Medical Research Council for 
an MRC Scholarship. 
References 
[l] Emtage, J. S., Lawson, D. E. M. and Kodicek, E. (1974) 
Biochem. J. 140,239-247. 
[2] Emtage, J. S., Lawson, D. E. M. and Kodicek, E. (1973) 
Nature 246,100-101. 
[3] Wasserman,R. H., Corradino, R. A. and Taylor, A. N. 
(1968) J. Biol. Chem. 243,3978-3986. 
(41 Jones, L. T. (1974) PhD Thesis, University of Cambridge. 
(51 Fullmer, C. S. and Wasserman, R. H. (1973) Biochim. 
Biophys. Acta 317,172-186. 
19 


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Investigation of cholecalciferol-dependent protein biosynthesis in the cytosol of the small intestine of the rachitic chick

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